The plasminogen activator (PA) system has an important role in controlling endogenous fibrosis and regulating the extracellular matrix (ECM) proteolysis relevant to tissue remodeling (Gabazza, E. C., et al., Lung, 177:253, 1999). The tissue-type PA (tPA) and urokinase-type PA (uPA) converts plasminogen to plasmin, which enhances proteolytic degradation of the ECM. An important mechanism in the regulation of PA activity is the inhibition of uPA or tPA by three major inhibitors, which are PAI-1, PAI-2, and PAI-3 (Kruithof, E. K., Enzyme, 40:113, 1998). Thus, as is well known, the plasminogen activator/plasmin system plays a critical role in fibrinolysis, cellular migration, and matrix remodeling. More specifically, Stefansson and Lawrence, Nature, 1996; 383:441–3, describes how PAI-1 blocks cell migration. Furthermore, Nar, et al., Journal of Molecular Biology, 2000; 297(3):683–95, describe the structure of PAI-1. Carmeliet, et al., J. Clin. Invest., 1993; 92:2746–2755, describe mice lacking sufficient PAI-1.
To elaborate, plasminogen is converted to its active form, plasmin, by serine proteases tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) (Sprengers E D, Kluft C. Plasminogen activator inhibitors. Blood 1987; 69: 381–7). Plasmin has a broad spectrum of proteolytic activities such as degradation of fibrin, activation of matrix metallo-proteases (MMPs) that degrade extracellular matrix (ECM) and play important roles in tissue remodeling. The t-PA activated plasminogen system is primarily responsible for degradation of fibrin. The balance between plasminogen activators (PA) and plasminogen activator inhibitor-1 (PAI-1) predominantly determines the plasma fibrinolytic activity (Rosenberg R D, Aird W C, 1999. Vascular-bed-specific hemostasis and hypercoagulable states. New England Journal of Medicine. 340:1555–1564). The u-PA activated plasminogen system functions in cell migration and tissue remodeling. The activation of plasminogen system is regulated either by inhibition of t-PA or u-PA by plasminogen activator inhibitor type-1 (PAI-1) (Francis R B Jr, Kawanishi D, Baruch T, Mahrer P, Rahimtoola S, Feinstein D I. Impaired fibrinolysis in coronary artery disease. Am Heart J 1988; 115:776–80) or by inhibition of plasmin by α2-antiplasmin (Booth N A. Natural inhibitors of fibrinolysis. In Bloom A L, Forbes C D, Thomas D P and Tuddenham E G D (eds) Haemostasis and Thrombosis, 3rd edin, pp699–717. Edinburg: Churchill Livingstone, 1994).
Plasma PAI-1 appears to mainly originate from the vascular endothelium, adipose tissue, and the liver (Loskutoff D J, N Y T, Sawdey M, Lawrence D., Journal of Cellular Biochemistry, 1986; 32:273–80; Samad F, Yamamoto K, Loskutoff D J, Journal of Clinical Investigation 1996; 97:37–46; Chomiki N, Henry M, Alessi M C, Anfosso F, Juhan-Vague I., Thrombosis & Haemostasis 1994; 72:44–53) and large quantities of which is stored by platelets and secreted upon platelet aggregation (Declerck P J, Alessi M C, Verstreken M, Kruithof E K, Juhan-Vague 1, Collen D., Blood 1988; 71:220–5). PAI-1 and t-PA exist in plasma in 4:1 molar ratio (Vaughan D E, Rouleau J-L, Ridker P M, Arnold J M O, Menapace F J, Pfeffer M A. Effects of ramipril on plasma fibrinolytic balance in patients with acute anterior myocardial infarction. Circulation 1997; 96:442–447) and PAI-1 in circulation has a T1/2 of approximately 5 minutes and is removed via a hepatic clearance mechanism (Vaughan D E, Declerck P J, Van Houtte E, De Mol M, Collen D., Circulation Research 1990; 67:1281–6).
Only a fraction of the secreted, active PAI-1 reacts with plasma t-PA, and forms inert, covalent complexes. Majority of PAI-1 in plasma and PAI-1 in the extracellular matrix of blood vessels binds to a 75 kD glycoprotein vitronectin (VN). The PAI-1-vitronectin complex may represent the physiologically relevant form of the inhibitor in the extracellular matrix (Keijer J, Ehrlich H J, Linders M, Preissner K T, Pannekoek H., Journal of Biol. Chem. 1991; 266:10700–7).
PAI-1 production is stimulated by a number of factors such as inflammatory cytokines, e.g. interleukin-I (IL-1) (Emeis J J, Kooistra, T., Journal of Experimental Medicine 1986; 163:1260–6) and tumor necrosis factor α (TNFα), transforming growth factor β (TGFβ) (Sawdey M, Podor T J, Loskutoff D J. Journal of Biological Chemistry 1989; 264:10396–401), epidermal growth factor (EGF), thrombin (Dichek D, Quertermous T. Blood 1989; 74:222–8) and insulin (Alessi M C, Juhan-Vague 1, Kooistra T, Declerck P J, Collen D., Thrombosis & Haemostasis 1988; 60:491–4). The infusion of endotoxin has also stimulated PAI-1 levels in plasma (Emeis J J, Kooistra. T., Journal of Experimental Medicine 1986; 163:1260–6; Colucci M, Paramo J A and Collen D., J. Clin Invest 1985; 75:818–24). Angiotensin II (Ang II) and angiotensin IV (Ang IV) also stimulate induction of PAI-1 transcription in vascular tissue in vitro and and in vivo (Vaughan D E, Lazos S A, Tong K., Journal of Clinical Investigation 1995; 95:995–1001; Feener E P, Northrup J M, Aiello L P, King G L., Journal of Clinical Investigation 1995; 95:1353–62).
The reactive center loop (RCL) of PAI-1 serves as the suicide inhibitory substrate for t-PA and u-PA by forming a covalent complex with PAs after its RCL is cleaved at 346Arg -347Met bond (P1-P1′) (Aertgeerts K, De Bondt H L, De Ranter C, Declerck P J., Journal of Structural Biology 1994; 113:23 9–45; Kruithof E K; Tran-Thang C, Ransijn A, Bachmann F., Blood 1984; 64:907–13). PAI-1 spontaneously acquires a thermodynamically more stable but functionally inactive latent form (Declerck P J, De Mol M, Alessi MC, et al., Journal of Biological Chemistry 1988; 263:15454–61). A series of amino acid substitutions (N150H, K154T, Q301P, Q315L and M354I) resulted in stabilization of reactive center loop of human PAI-1 in the active conformation (referred to as PAI-1-stab) and extended the T1/2 of the enzyme from 2.5 hrs to >145 hrs at 37° C. in vitro (M. B. Berkenpas, D. A. Lawrence and D. Ginsburg, EMBO J. (1995) 14: 2969–2977). Clinical evidence linking PAI-1 with arterial and venous thrombosis stresses physiological importance of PAI-1 (Wiman B, Ljungberg B, Chmielewska J, Urden G, Blomback M, Johnsson H., J Lab Clin Med 1985; 105:265–70; Auwerx, J., Bouillon R, Collen D, Geboers, J., Arteriosclerosis 1988; 8:68–72; Margaglione M, Di Minno G, Grandone E, et al., Arterioscler Thromb 1994;14:1741–5; Thogersen A M, Jansson J H, Boman K, et al., Circulation 1998; 98:2241–7; Juhan-Vague 1, Valadier J, Alessi M C, et al., Thrombosis & Haemostasis 1987; 57:67–72).
Despite the above-described efforts, there remains a need in the art for further characterization of the biological role of PAI-1. An animal model to facilitate such characterization is also needed. The present invention addresses these and other needs in the art.